Anti-neoplastic viral agents

ABSTRACT

A viral DNA construct, and virus encoded thereby, is provided having one or more tumor specific transcription factor binding sites in place of one or more wild type transcription factor binding sites operatively positioned in the promoter region which controls expression of early genes responsible for viral nucleic acid replication. Preferred constructs place the tumor specific transcription factor binding sites in operative relation to DNA polymerase, DNA terminal protein and/or DNA binding protein. Compositions and constructs contained therein are provided, particularly for use in therapy. Methods of treating patients for neoplasms are also provided.

This application is a continuation of Application No. PCT/GB00/01142,filed Mar. 24, 2000, the entire content of which is hereby incorporatedby reference in this application.

The present invention provides viral agents that have application in thetreatment of neoplasms such as tumours, particularly tumours derivedfrom colon cells, more particularly liver tumours that are metastases ofcolon cell primary tumours. Still more particularly are providedreplication efficient adenovirus constructs that selectively replicatein response to transcription activators present in tumour cells, thesefactors being present either exclusively or at elevated levels in tumourcells as compared to other cells, and thus which lead to tumour celldeath and cell lysis.

By injecting these viral agents locally into the liver it is possible totreat liver metastases; which are a major cause of morbidity in coloncancer patients. Applications beyond this, e.g. to other sites and othertumours, such as colorectal cancers and melanomas, are also provided.

Colon cancer presents with locally advanced or metastatic disease in themajority of patients. Most patients are left with liver metastases asthe only site of disease after resection of the primary tumour. Partialliver resection only cures about 10% of patients, while in patients withmultiple metastases in both liver lobes resection is not feasible andloco-regional or systemic treatment with chemotherapy is indicated(Labianca et al., 1997). Systemic chemotherapy with 5-fluorouracil andleucovorin or irinotecan will produce response rates of only 20%(Cunningham et al., 1998; Stupp et al., 1998).

Locoregional chemotherapy of the liver has been explored for over 15years. Most liver metastases are supplied with blood by the hepaticartery, so intra-arterial hepatic chemotherapy (IAHC) allows for muchhigher exposure of the metastases to cytotoxic drugs. The highextraction rate of normal liver decreases the systemic drugconcentration resulting in less toxicity with IAHC having been shownrepeatedly to give response rates over 60% (Kemeny et al., 1987; Kemenyet al., 1992; Patt and Mavligit, 1991).

Specific defects in tumour cells make it possible to devise rationalstrategies for targeting tumour cells without harming normal cells. Withthe exception of anti-angiogenic therapy, this usually requiresintroduction of exogenous DNA into tumour cells, and the most efficientway to do this is with viruses. For example, U.S. Pat. No. 5,698,443describes a tumour specific adenovirus that is targeted at prostatecancer cells. This virus utilises a prostate specific enhancer sequencedriving the viral E1 genes and the patent suggests its use to expresstoxins specifically in the target cell.

Viruses which replicate selectively in tumour cells have great potentialfor gene therapy for cancer. In principle, selectively replicatingcytotoxic viruses can spread progressively through a tumour until all ofits cells are destroyed. This overcomes the need to infect all tumourcells at the time the virus is injected, which is a major limitation toconventional replacement gene therapy, because in principle virus goeson being produced, lysing cells on release of new virus, until no tumourcells remain. An important fundamental distinction in cancer genetherapy is thus between single hit approaches, using non-replicatingviruses, and multiple hit approaches, using replicating viruses.

Single hit approaches work by directly transducing tumour cells withtoxic genes; ignoring bystander effects, one virus particle kills onecell. Examples include restoration of tumour suppressor gene expressionand conditional expression of toxins using tumour-specific promoters.For single hit approaches the amount of virus injected is an importantlimiting factor. Multiple hit approaches circumvent this limitationeither by provoking an immune reaction against tumour cells, or by usingviruses that replicate within the tumour. Since the majority of tumourcells are not killed by the injected virus itself, the amount of virusinjected should not be an important factor limiting the therapeuticresponse.

Classic gene replacement therapy has been performed with retrovirusesexpressing p53 (Roth et al., 1996). Since p53 can be converted to itsoncogenic form by mutations at over 500 sites in the open reading frame(Flaman et al., 1994), retroviral replication will convert at least 1%of the transduced p53 to such undesired mutant form. This means thateach patient received around 25 million infectious units of virusexpressing mutant p53 (Estreicher and Iggo, 1996).

A more promising approach expressing wild type p53 using an adenovirus,Ad-CMV-p53, has been demonstrated clinically in head and neck cancer andis currently under investigation in lung, colon and liver cancer(Clayman et al., 1998). Adenoviruses are relatively stable, can beproduced at high titres, and can infect both quiescent and dividingcells of many different types. Overall, Ad-CMV-p53 appears exceptionallynon-toxic but probably ineffective as a single agent; hence, there is aplace for more aggressive second generation viruses.

Further target specific defects are mutations of p16, cdk4, cyclin D orRb (Bartek et al., 1997) in the retinoblastoma pathway which cause lossof G1/S control and essentially all tumours have these. The onlysignificant exception is colon cancer, where mutations in the Rb pathwayitself are rare. The net result of these defects is increased E2Factivity, which means that tumours can be selectively targeted byviruses expressing toxic genes from E2F-regulated promoters. This hasbeen demonstrated using an adenovirus expressing the HSV thymidinekinase gene from such a promoter (Parr et al., 1997); cells containingRb-pathway mutations express tk and can be killed by ganciclovir. Suchan approach relies on an increase in the activity of specifictranscription factors in tumour cells.

The rational basis for tumour targeting is better understood fornon-replicating E2F-targeting viruses than it is for p53, but both arestill single hit approaches and it is very difficult to see how they canever be used for more than treatment of local disease. The tumour burdenin late stage disease is around 10¹² cells, so while at an effectivemultiplicity of infection of one treatment would be feasible, inpractice biodistribution and receptor problems mean that many orders ofmagnitude higher multiplicities are required.

One elegant way to circumvent this limitation is to recruit the immunesystem to kill the tumour cells. The role of the gene therapy virus issimply to provoke or reinforce the immune response. There is abundantevidence that tumours express new antigens, but in cancer patients theimmune system has clearly failed to prevent tumour formation. Manycurrently attempted techniques target single antigens, eg production ofcytotoxic T cells against MAGE antigens in melanoma, but the goal fortherapy must be to induce a simultaneous response against multipledifferent antigens, because genetic instability in tumours meanstargeting of single antigens is unlikely to produce lasting responsesbecause the number of tumour cells exceeds the mutation rate. Henceacquired resistance to immunotherapy is common.

An attractive solution to the single hit problem is to produce viruswithin the tumour. This can be achieved by injecting retroviral producercell lines into the tumour bed, a strategy currently being tested in aclinical trial for glioblastoma (reviewed by Roth and Cristiano, 1997).This is an elegant but limited approach as it relies on immune privilegein the CNS to avoid immediate rejection of the grafted cells, and tumourtargeting depends on the fact that retroviruses do not infectnon-dividing normal brain cells. It falls short of the goal of tumourcell-specific viral replication because the viruses produced are notthemselves replication competent and virus production is dependent onsurvival of the packaging cells rather than the presence of tumourcells.

The prototype tumour selective virus is a defective adenovirus lackingthe E1B 55K gene (d1 1520ONYX 015, Bischoff et al., 1996). In normaladenoviruses 55K inactivates p53, hence it should not be required incells where p53 is mutant. In practice, many cells containing wild typep53 are killed by the virus (Heise et al., 1997). The present inventorshave tested this in H1299 p53-null lung carcinoma cells containing wildtype p53 under a tetracycline-regulated promoter and found that dl 1520replicates as well in the presence as in the absence of wild type p53.Besides targeting p53, E1B 55K is required for selective viral RNAexport (Shenk, 1996) and it is not immediately obvious how loss of p53could substitute for this function. At present there is no convincingevidence that dl 1520 targets p53 defects (Goodrum 1997, Goodrum 1998,Hall 1998, Rothman 1998, Turnell 1999).

As with p53-expressing viruses, combination therapy with chemotherapyand d1 1520 gives better results both in vitro and in xenografts (Heiseet al., 1997). In principle, the virus should undergo multiple rounds ofreplication until there are no tumour cells remaining and since eachinfected cell produces 10³ to 10⁴ new virus particles, the amount ofinput virus should not be limiting. In practice, the required amount ofd1 1520 virus injected is comparable for therapy with Ad-CMV-p53. Thismeans that the virus is not performing as expected for a replicatingvirus with the reasons for this again probably quite complex.Adenoviruses normally produce superficial mucosal infections which arespread by droplets containing infected cells. Infected cells retainprogeny virus. Lack of effective virus release from lysed cells willmilitate against the production of deep, spreading infection, which isthe goal if virus is to penetrate to all parts of the tumour.

Rational targeting of E2F defects is complicated by the fact that aspart of its life cycle the adenovirus already produces proteins (E1A andE4 orf 6/7) which target E2F. Since E1A and orf 6/7 are multifunctionalproteins the effect of E1A and orf 6/7 mutations is complex andunpredictable.

In addition to E2F and p53, there are four transcription factors whoseactivity is known to increase in tumours. They are Tcf4, RBPJκ andGli-1, representing the endpoints of the wnt, notch and hedgehog signaltransduction pathways (Dahmane et al., 1997; Jarriault et al., 1995; vande Wetering et al., 1997) and HIF1 alpha, which is stabilised bymutations in the Von Hippel Lindau tumour suppressor gene (Maxwell et al1999). Mutations in APC or β-catenin are universal defects in coloncancer (Korinek et al., 1997; Morin et al., 1997); but they also occurat lower frequency in other tumours, such as melanoma (Rubinfeld et al.,1997). Such mutations lead to increased Tcf activity in affected cells.The hedgehog pathway is activated by mutations in the patched andsmoothened proteins in basal cell cancer (Stone et al., 1996; Xie etal., 1998). Notch mutations occur in some leukaemias (Ellisen et al.,1991). Telomerase activation is one of the hallmarks of cancer (HanahanD. and Weinberg RA. The hallmarks of cancer. Cell. 100, 57-70, 2000) andresults from increased activity of the telomerase promoter, although themechanism is unknown. According to Cong Y S et al (1999, HMG 8, 137-42)the elements responsible for promoter activity are contained within aregion extending from 330 bp upstream of the ATG to the second exon ofthe gene and thus this sequence is a further suitable promoter sequencefor use in the viral constructs and viruses of the invention.

The present inventors have now designed and produced viral DNAconstructs, and replicating viruses encoded thereby, preferably in theform of recombinant viral constructs and viruses, which rationallytarget known causal oncogenic transcription defects in tumours by usingthese to control transcription of one or more early viral genes encodingfor proteins which are mechanistically directly involved in viralnucleic acid replication (for examples of such genes see eg.DePamphilis, M L, Concepts in Eukaryotic DNA Replication pp 481-484 and488-491, 1999 Cold Spring Harbor Laboratory Press, New York which isincorporated herein by reference). Preferably these viral genes areselected from the group consisting of polymerase, primase, nuclease,helicase, ligase, terminal protein and nucleic acid binding proteingenes. Preferred enzymatic products of five of these genes areclassified in the following Enzyme Commission classifications:polymerase (EC 2.7.7.7), primase (EC 2.7.7.6), nuclease (EC 3.1.11-25),helicase (EC 3.6.1.3) and ligase (EC 6.5.1.1-2). These classificationsmay vary, for example, in parvoviruses, eg. Adenoassociatedviruses(AAV), they include NS1/Rep proteins, which have nuclease and helicaseactivities essential for replication In the preferred constructs andviruses produced by the inventors these genes are particularly the viralDNA polymerase, viral DNA terminal protein and/or viral DNA bindingprotein genes. Preferred viruses are those having E1, E2 and E3 viraltranscription units, such as adenoviruses. It is particularly preferredthat at least the E2 transcription unit of the virus is altered suchthat it is made responsive to factors that are present at increasedlevels or increased activity, or are only present in, a target tumourcell. Rittner et al (J. Virol (1997) p 3307-3311) teach that it is notpossible to manipulate the E2 promoters close to the cap sites becauseof the overlap with the major late open reading frame L4. The presentinventors however find that this manipulation is indeed possible withadvantageous results.

It has thus been determined that by controlling nucleic acid replicationcapability, rather than factors such as RNA export capability ortoxicity due to insertion of recombinant genes, the inventors canprovide selectivity of cytotoxic effect to tumour cells.

Most importantly and advantageously, the present inventors have madepossible to target tumour cells with a virus encoding only wild typeviral proteins, whose expression is specifically regulated bytranscription factors preferentially or exclusively activated in tumourcells. Preferred virus encodes a full set of wild type proteins. Suchvirus can be used to better effect than prior art viral agents whichhave relied on mutation of viral proteins or targeting of cells of aparticular tissue origin, eg. prostate.

Thus in a first aspect of the present invention there is provided aviral DNA construct encoding for a virus that is capable of replicationin a human or animal tumour cell type and causing tumour cells of thattype to die characterised in that the construct comprises one or moreselected transcription factor binding sites together with, andoperatively positioned such as to promote expression of, open readingframes encoding early viral proteins, the protein products of thosereading frames being mechanistically directly involved in viralconstruct nucleic acid replication wherein the selected transcriptionfactor binding sites are for a transcription factor the level oractivity of which is increased in a human or animal tumour cell relativeto that of a normal human or animal cell of the same type.

Preferred constructs of the invention have a nucleic acid sequencecorresponding to that of a wild type virus sequence characterised inthat it has one or more wild type transcription factor binding sitesreplaced by one or more selected transcription factor binding sites,these sites being operatively positioned in the promoter region whichcontrols expression of early genes such as to promote expression of theopen reading frame of the gene, the protein products of those genesbeing mechanistically directly involved in viral nucleic acidreplication

wherein the selected transcription factor binding sites are for atranscription factor the level or activity of which is increased in ahuman or animal tumour cell relative to that of a normal human or animalcell of the same type.

Preferably the viral DNA construct is characterised in that the siteswhich replace the wild type transcription factor sites controllingexpression of said early genes are for a transcription factor whoseactivity or level is specifically increased by causal oncogenicmutations.

While the controlled open reading frames or genes may be one or more ofthe viral polymerase, primase, nuclease, helicase and ligase, preferablythey are one or more of the DNA polymerase, DNA terminal protein and/orDNA binding protein. More preferably the construct or virus is such thatit has E1, E2 and E3 regions and the tumour specific transcriptionfactor binding site replaces one or more wild type E2 transcriptionfactor binding sites.

Preferably the viral DNA construct is characterised in that it encodes afunctional viral RNA export capacity. For adenovirus this is encoded inthe E1 and E4 regions, particularly the E1B 55K and E4 orf 6 genes. Thuspreferably the encoded virus is of wild type with respect to expressionof these genes in tumour cells. Most preferably the E1B 55K and E4 orf 6open reading frames are functional and/or intact where present in thecorresponding wild type virus.

It will be realised by those skilled in the art that any virus which ispotentially cytotoxic to tumour cells may be employed in producing viralconstructs of the present invention. Particularly examples may includeadenovirus, lentivirus, polyoma virus, vaccinia virus, herpesvirus andparvovirus. Preferably the virus is an adenovirus, more preferably anadenovirus that is of high specificity for a target tumour cell type,eg. for a colon tumour type.

Preferred colon tumour specific adenoviruses are encoded by viral DNAconstructs corresponding to the DNA sequence of Ad5 or one or more ofthe enteric adenoviruses Ad40 and Ad41 modified as described above. Ad40and Ad41, which are available from ATCC, are selective for colon cellsand one important difference to Ad5 is that there is an additional fibreprotein. The fibre protein binds to the cell surface receptor, calledthe coxsackie-adeno receptor or CAR for Ad5. Colon cells have less CARthan lung cells which Ad5 is adapted to infect. Ad40 and Ad41 have twofibre proteins, with the possibility being that they may use twodifferent receptors. The expected form of resistance to virus therapy isloss of the receptor, which obviously prevents infection. Geneticinstability in tumours means this will happen at some reasonablefrequency; about 1 in 100 million cells, a mutation rate of 1 in 10⁸. Ifyou have to delete two receptors you multiply the probabilities; ie.loss of both will occur in 1 in 10¹⁶ cells. A tumour contains between10⁹ and 10¹² cells. Hence resistance is less likely to develop if avirus uses more than one receptor. One fibre protein in Ad40 and 41 usesCAR whilst the receptor used by the other is as yet unknown.

Advantageously the use of the constructs of the invention, particularlyin the form of viruses encoded thereby, to treat liver metastasis isrelatively non-toxic compared to chemotherapy, providing good spread ofvirus within the liver aided by effective replication.

Preferred viral constructs of the invention are derived from adenovirusor parvovirus genomic DNA, more preferably adenovirus genomic DNA, andare mutated such that transcription of essential viral genes encoded bythe E2 viral transcription unit is made dependent on the presence ofoncogenic mutations in tumour cells. Preferably only cells containingthese oncogenic mutations can activate transcription of the viral E2genes. Since the E2 unit encodes the viral DNA polymerase, DNA terminalprotein and DNA binding protein, the virus can only replicate in tumourcells. It is preferred that the E2 early promoter transcription factorbinding site is replaced by the tumour cell specific transcriptionfactor binding site.

Preferred tumour specific transcription factor binding sites that areused in place of wild type sites are those described above as Tcf-4,HIF1alpha, RBPJκ and Gli-1 sites, and a fragment of the telomerasepromoter conferring tumour-specific transcription. A most preferredtranscription factor binding site is that which binds Tcf-4, such asdescribed by Vogelstein et al in U.S. Pat. No. 5,851,775 and isresponsive to the heterodimeric β-catenin/Tcf-4 transcription factor. Assuch the transciption factor binding site increases transcription ofgenes in response to increased β-catenin levels caused by APC orβ-catenin mutations. The telomerase promoter is described by Wu K J. etal (1999, Nat Genet 21, 220-4) and Cong Y S. et al (1999 HumMol Genet 8,137-42). A further preferred binding site is that of HIF1alpha, asdescribed by Maxwell P H. et al, (1999 Nature 399, 271-5). One may use aHIF1alpha-regulated virus to target the hypoxic regions of tumours,involving no mutation of the pathway as this is the normal physiologicalresponse to hypoxia, or the same virus may be used to target cells withVHL mutations either in the familial VHL cancer syndrome, or in sporadicrenal cell carcinomas, which also have VHL mutations. A retrovirus usingthe HIF promoter to target hypoxia in ischemia has already beendescribed by Boast K. et al (1999 Hum Gene Ther 10, 2197-208).

Particularly the inventors have now provided viral DNA constructs, andviruses encoded thereby, which contain the Tcf transcription factorbinding sites referred to above in operational relationship with theearly gene open reading frames described above, particularly in place ofwild type transcription factor binding sites in the E2 promoter andshown that these are selective for tumour cells containing oncogenic APCand β-catenin mutations. Tcf-4 and its heterodimer bind to a sitedesignated Tcf herein. Preferred such replacement sites are single ormultiples of the Tcf binding sequence, eg. containing 2 to 20, morepreferably 2 to 6, most conveniently, 2, 3 or 4 Tcf sites.

Particular Tcf sites are of consensus sequence (A/T)(A/T)CAA(A/T)GG, seeRoose, J., and Clevers, H. (1999 Biochim Biophys Acta 1424, M23-37), butare more preferably as shown in the examples herein.

More preferred viral constructs and viruses of the invention are thosehaving E3 domains and are characterised in that they have mutations toone or more residues in the NF1, NFκB, AP1 and/or ATF regions of the E3promoter, more preferably those mutations which reduce E2 genetranscription caused by E3 promoter activity. The present inventors haveparticularly provided silent mutations, these being such as not to alterthe predicted protein sequence of any viral protein, which alter theactivity of key viral promoters.

NFκB is strongly induced in regenerating liver cells, ie. hepatocytes(see Brenner et al J. Clin. Invest. 101 p802-811). Liver regeneration tofill the space vacated by the tumour is likely to occur followingsuccessful treatment of metastases. In addition, if one wishes to treathepatoma, which arise on a background of dividing normal liver cells,then destroying the NFκB site is potentially advantageous.

In a preferred embodiment of the first aspect of the present inventionthe inventors have replaced a short region in the E2 early promoter,which is not overlapped by coding sequence, with multiple Tcf bindingsites, more preferably 3 or 4 such sites, and most preferably 4 sites.One resulting preferred viral construct and encoded virus are referredto herein as Ad-Tcf3, having 3 such sites, and the virus expresses E2gene products and replicates better in colon than in lung tumour cells(see Examples). This shows that mutations of the type described canmodify the activity of the E2 promoter in the desired way withoutuntoward effects on other aspects of the viral life cycle. This is notobvious a priori because, as well as encoding the E2 promoter, thisregion is transcribed and retained in the 5′-untranslated region of thepVIII protein RNA, it is transcribed but spliced out of the L5 late RNAand it forms part of the 3′-untranslated region of the 33k protein RNA.

Although Ad-Tcf3 replicates better in colon than lung tumour cells, thedifference is only around ten-fold. LGC, a virus of the inventioncombining the E2 mutations in Ad-Tcf with an E1B 55K deletion, showsaround 1000-fold selectivity for colon cancer cells. This demonstratesthat E2 promoter activity can be made limiting for viral replication,because the identical virus with the normal E2 promoter (LGM) shows nospecificity for colon cells. For a colon-targeting strategy this is animportant result because it means a colon-specific virus can be made bytitrating the E2 promoter activity.

The probable explanation for the selectivity of LGC is that the E1B 55kprotein is required for nuclear export of late viral RNAs, including theE2 DBP late RNA, and that the E2 RNA export defect in E1B 55K-deficientviruses can be overcome by increasing E2 RNA production by inserting Tcfsites in place of the normal transcription factor binding sites in theE2 promoter.

One method for increasing the specificity of the AdTcf3, and similar3xTcf driven E2 viruses of the invention for colon tumours is to reduceits E2 promoter activity in non-colon cells. One possible way to do thisis to alter the number of Tcf sites. Reduction in the number of Tcfsites to two could reduce non-specific leakiness, but since Tcfpromoters are actively repressed in non-colon cells by groucho (Fisherand Caudy, 1998) and acetylation (Waltzer 1998) it is more likely thatincreasing the sites will give a more tightly regulated promoter as themore sites there are the more repressor will be bound in non-coloncells. E1A normally activates the E2 promoter through the ATF site. Inthe absence of such targeting E1A represses promoters, eg. by chelatingp300/CBP. Since the ATF site is deleted in the Tcf-mutant E2 promoter,E1A produced by the virus should reduce general leakiness of the mutantE2 promoter in all cell types. The E3 promoter is back-to-back with theE2 promoter and the distinction between them is defined but functionallyarbitrary. Hence further reduction of the activity of the mutant E2promoter is possible by modifying or deleting transcription factorbinding sites in the E3-promoter. Since the E3 promoter lies in codingsequence it cannot just be deleted. Instead the inventors have providedup to 16 silent substitutions changing critical residues in known NF1,NFκB, AP1 and ATF sites (Hurst and Jones, 1987, Genes Dev 1, 1132-46,incorporated herein by reference).

Further viral constructs of the present invention may be provided bymodifying the E2-late promoter of adenoviruses. The E2-early promotercontrols transcription of DNA polymerase (pol), DNA binding protein(DBP) and preterminal protein (pTP). By mutating the E2 late promoter itis possible to have a similar effect, ie. at least in part, to the E1Bdeletion because E1B deletion reduces export of DBP RNA expressed fromthe E2 late promoter. DBP is required stoichiometrically for DNAreplication, so reducing DBP production in normal cells is desirable.Since the E2 late promoter lies in 100k protein coding sequence itcannot just be deleted. Instead the inventors have determined that itcan inactivated with silent mutations changing critical residues inknown transcription factor binding sites.

Particular transcription factor binding sites in the E2 late promoterwere identified by DNase I footprinting (marked I-IV in FIG. 4 herein;Goding et al, 1987, NAR 15, 7761-7780). The most important is a CCAATbox lying in footprint II. Mutation of this CCAAT box reduces E2 latepromoter activity 100-fold in CAT assays (Bhat et al, 1987, EMBO J,6,2045-2052). One such mutation changes the marked CCAAT box sequenceGAC CAA TCC to GAT CAG TCC. (see FIG. 4 below). This is designed toabolish binding of CCAAT box binding factors without changing the 100kprotein sequence. Additional silent mutations in the other footprintscan be used to reduce activity further.

An alternative or additional mutation possible is to regulate expressionof E1B transcription by mutating the E1B promoter. This has been shownto reduce virus replication using a virus in which a prostate-specificpromoter was used to regulate E1B transcription (Yu, D. C., et al 1999Cancer Research 59, 1498-504). A further advantage of regulating E1B 55Kexpression in a tumour-specific manner would be that the risk ofinflammatory damage to normal tissue would be reduced (Ginsberg, H. S.,et al 199 PNAS 96, 10409-11). The inventors have produced viruses withTcf sites replacing the E1B promoter Sp1 site to test this proposition.

Further embodiments include the following possible modifications. Tofurther restrict replication one can insert further tumour specific, eg.Tcf, sites in the E1A promoter. To achieve regulation by inserting shortoligonucleotides, one must delete the existing regulatory sequences inthe E1A promoter. This requires simultaneous mutation of both invertedterminal repeats and transfer of the packaging signal elsewhere, eg. tothe E4 region. In contrast with, for example, the Calydon viruses, thedesign of the present inventors viruses means that, despite retaining afull complement of adenoviral genes, spare packaging capacity isavailable, which can be used to express conditional toxins, such as theprodrug-activating enzyme HSV thymidine kinase (tk). This could beexpressed for example from the E3 promoter, whose activity is regulatedin some of the viruses, to provide an additional level of tumourtargeting. Alternatively, it could be expressed from a constitutivepromoter to act as a safety feature, since ganciclovir would then beable to kill the virus. Constitutive tk expression in an E1B-deficientvirus also increases the tumour killing effect, albeit at the expense ofreplication (Wildner, O., et al 1999 Gene Therapy 6, 57-62). Analternative prodrug-activating enzyme to express would be cytosinedeaminase (Crystal, R. G., et al 1997 Hum Gene Ther 8, 985-1001), whichconverts 5FC to 5FU. This has advantage because 5FU is one of the fewdrugs active on liver metastases, the intended therapeutic target, butproduces biliary sclerosis in some patients.

The amino-terminus of E1A contains a region of E1A that binds p300, ahistone acetylase which functions as a general transcription factor. Oneway E1A activates genes is by bringing p300 to the promoter. To do thisE1A binds transcription factors like ATF. Hence E1A activates promotersthat contain ATF sites. Virus vMB13 herein retains the ATF site in theE3 promoter providing advantage in this respect. The problem is that ifa promoter does not have an ATF site, E1A will repress it by bindingp300. This is what happens with p53, for example: E1A blocksp53-dependent transcription in a manner that requires the p300 bindingsite in E1A. Tcf repression by E1A is a possibility in some cell lines,so mutation of the E1A p300-binding site may be preferred for suchtreatment.

The present inventors see a difference between vMB13 and vMB14 in HCT116 cells, where the only difference between the two viruses is in theATF site in the E3 promoter. Thus mutation of the E1A p300-binding sitein vMB14 might be advantageous. Alternatively, the difference could bedue to direct activation of the ATF site because Xu L et al (2000, GenesDev 14, 585-595) report that ATF/CREB sites can be activated by wntsignals, although the mechanism is unknown.

Having produced a virus with one or more levels of regulation to preventor terminate replication in normal cells, it is further preferred andadvantageous to improve the efficiency of infection at the level ofreceptor binding. The normal cellular receptor for adenovirus, CAR, ispoorly expressed on some colon tumour cells. Addition of a number oflysine residues, eg 15 to 25, more preferably about 20, to the end ofthe adeno fibre protein (the natural CAR ligand) allows the virus to useheparin sulphate glycoproteins as receptor, resulting in more efficientinfection of a much wider range of cells. This has been shown toincrease the cytopathic effect and xenograft cure rate of E1B-deficientviruses (Shinoura, H., et al 1999 Cancer Res 59, 3411-3416 incorporatedherein by reference).

A n alternative strategy is to incorporate the cDNA encoding for Ad40and/or Ad4l fibres into the construct of the invention as describedabove. The EMBL and Genbank databases list scuh sequences and they arefurther described in Kidd et al Virology (1989) 172(1), 134-144;Pieniazek et al Nucleic Acids Res. (1989) November 25; 17-20, 9474;Davison et al J. Mol. Biol (1993) 234(4) 1308-16; Kidd et al Virology(1990) 179(1) p139-150; all of which are incorporated herein byreference.

In a second aspect of the invention there is provided the viral DNAconstruct of the invention, particularly in the form of a virus encodedthereby, for use in therapy, particularly in therapy of patients havingneoplasms, eg. malignant tumours, particularly colorectal tumours andmost particularly colorectal metastases. Most preferably the therapy isfor liver tumours that are metastases of colorectal tumours.

In a third aspect there is provided the use of a viral DNA construct ofthe invention, particularly in the form of a virus encoded thereby, inthe manufacture of a medicament for the treatment of neoplasms, eg.malignant tumours, particularly colorectal tumours and most particularlycolorectal metastases. Most preferably the treatment is for livertumours that are metastases of colorectal tumours.

In a fourth aspect of the invention there are provided compositionscomprising the viral DNA construct of the invention, particularly in theform of a virus encoded thereby, together with a physiologicallyacceptable carrier. Such carrier is typically sterile and pyrogen freeand thus the composition is sterile and pyrogen free with the exceptionof the presence of the viral construct component or its encoded virus.Typically the carrier will be a physiologically acceptable saline.

In a fifth aspect of the invention there is provided a method ofmanufacture of the viral DNA construct of the invention, particularly inthe form of a virus encoded thereby comprising transforming a viralgenomic DNA, particularly of an adenovirus, having wild typetranscription factor binding sites, particularly as defined for thefirst aspect, controlling transcription of genes the protein products ofwhich are directly mechanistically involved in viral nucleic acidreplication, such as to operationally replace these sites by tumourspecific transcription factor binding sites, particularly replacing themby Tcf transcription factor binding sites. Operational replacement mayinvolve partial or complete deletion of the wild type site. Preferablythe transformation inserts two or more, more preferably 3 or 4, Tcf-4transcription factor binding sites. More preferably the transformationintroduces additional mutations to one or more residues in the NF1,NFκB, AP1 and/or ATF binding sites in the E3 promoter region of theviral genome. Such mutations should preferably eliminate interferencewith E2 activity by E3 and reduce expression of E2 promoter-driven genesin normal cells and non-colon cells. Reciprocally, it preferablyreplaces normal regulation of E3 with regulation by Tcf bound to thenearby E2 promoter.

Traditional methods for modifying adenovirus require in vivoreconstitution of the viral genome by homologous recombination, followedby multiple rounds of plaque purification. The reason for this is thedifficulty of manipulating the 36kb adenovirus genome using traditionalcloning techniques. Newer approaches have been developed whichcircumvent this problem, particularly for E1-replacement vectors. TheTransgene and Vogelstein groups use gap repair in bacteria to modify thevirus (Chartier et al., 1996; He et al., 1998). This requires theconstruction of large vectors which are specific for each region to bemodified. Since these vectors are available for E1-replacement, theseapproaches are very attractive for construction of simple adenoviralexpression vectors. Ketner developed a yeast-based system where theadenoviral genome is cloned in a YAC and modified by two step genereplacement (Ketner et al., 1994). The advantage of the YAC approach isthat only very small pieces of viral DNA need ever be manipulated usingconventional recombinant DNA techniques. Conveniently, a few hundredbase pairs on either side of the region to be modified are provided andon one side there should be a unique restriction site, but since theplasmid is very small this is not a problem. The disadvantage of theKetner approach is that the yield of YAC DNA is low.

The present inventors have combined the bacterial and yeast approaches.Specifically, they clone the viral genome by gap repair in a circularYAC/BAC in yeast, modify it by two step gene replacement, then transferit to bacteria for production of large amounts of viral genomic DNA. Thelatter step is useful because it permits direct sequencing of themodified genome before it is converted into virus, and the efficiency ofvirus production is high because large amounts of genomic DNA areavailable. They use a BAC origin to avoid rearrangement of the viralgenome in bacteria. Although this approach has more steps, it combinesall of the advantages and none of the disadvantages of the purebacterial or yeast techniques.

Although it can be used to make E1-replacement viruses, and theinventors have constructed YAC/BACs allowing cycloheximide selection ofdesired recombinants in the yeast excision step to simplify this task,the main strength of the approach is that it allows introduction ofmutations at will throughout the viral genome. Further details of theYAC/BAC are provided by the inventors as their contribution to Gagnebinet al (1999) Gene Therapy 6, 1742-1750) which is incorporated herein byreference. :Sequential modification at multiple different sites is alsopossible without having to handle large DNA intermediates in vitro.

The adenovirus strain to be mutated using the method of the invention ispreferably a wild type adenovirus. Conveniently adenovirus 5 (Ad 5) isused, as is available from ATCC as VR5. The viral genome is preferablycompletely wild type outside the regions modified by the method, but maybe used to deliver tumour specific toxic heterologous genes, eg. p53 orgenes encoding prodrug-activating enzymes such as thymidine kinase whichallows cell destruction by ganciclovir. However, the method is alsoconveniently applied using viral genomic DNA from adenovirus types withimproved tissue tropisms (eg. Ad40 and Ad41).

In a sixth aspect of the present invention there is provided a methodfor treating a patient suffering from neoplasms wherein a viral DNAconstruct of the invention, particularly in the form of a virus encodedthereby, is caused to infect tissues of the patient, including orrestricted to those of the neoplasm, and allowed to replicate such thatneoplasm cells are caused to be killed.

The present invention further attempts to improve current intra-arterialhepatic chemotherapy by prior administration of a colon-targetingreplicating adenovirus. DNA damaging and antimetabolic chemotherapy isknown to sensitise tumour cells to another replicating adenovirus inanimal models (Heise et al., 1997). For example, during the first cyclethe present recombinant adenovirus can be administered alone, in orderto determine toxicity and safety. For the second and subsequent cyclesrecombinant adenovirus can be administered with concomitantchemotherapy. Safety and efficacy is preferably evaluated and thencompared to the first cycle response, the patient acting as his or herown control.

Route of administration may vary according to the patients needs and maybe by any of the routes described for similar viruses such as describedin U.S. Pat. No. 5,698,443 column 6, incorporated herein by reference.Suitable doses for replicating viruses of the invention are in theorycapable of being very low. For example they may be of the order of from10² to 10¹³, more preferably 10⁴ to 10¹¹, with multiplicities ofinfection generally in the range 0.001 to 100.

For treatment a hepatic artery catheter, eg a port-a-cath, is preferablyimplanted. This procedure is well established, and hepatic catheters areregularly placed for local hepatic chemotherapy for ocular melanoma andcolon cancer patients. A baseline biopsy may be taken during surgery.

A typical therapy regime might comprise the following:

Cycle 1: adenovirus construct administration diluted in 100 ml salinethrough the hepatic artery catheter, on days 1, 2 and 3.

Cycle 2 (day 29): adenovirus construct administration on days 1, 2, and3 with concomitant administration of FUDR 0.3 mg/kg/d as continuousinfusion for 14 days, via a standard portable infusion pump (e.g.Pharmacia or Melody), repeated every 4 weeks.

Toxicity of viral agent, and thus suitable dose, may be determined byStandard phase I dose escalation of the viral inoculum in a cohort ofthree patients. If grade III/IV toxicity occurs in one patient,enrolment is continued at the current dose level for a total of sixpatients. Grade III/V toxicity in ≧50% of the patients determines doselimiting toxicity (DLT), and the dose level below is considered themaximally tolerated dose (MTD) and may be further explored in phase IItrials.

It will be realised that GMP grade virus is used where regulatoryapproval is required.

It will be realised by those skilled in the art that the administrationof therapeutic adenoviruses may be accompanied by inflammation and orother adverse immunological event which can be associated with eg.cytokine release. Some viruses according to the invention may alsoprovoke this, particularly if E1B activity is not attenuated. It willfurther be realised that such viruses may have advantageous anti-tumouractivity over at least some of those lacking this adverse effect. Inthis event it is appropriate that an immuno-suppressive,anti-inflammatory or otherwise anti-cytokine medication is administeredin conjunction with the virus, eg, pre-, post- or during viraladminstration. Typical of such medicaments are steroids, eg,prednisolone or dexamethasone, or anti-TNF agents such as anti-TNFantibodies or soluble TNF receptor, with suitable dosage regimes beingsimilar to those used in autoimmune therapies. For example, see doses ofsteroid given for treating rheumatoid arthritis (see WO93/07899) ormultiple sclerosis (WO93/10817), both of which in so far as they have USequivalent applications are incorporated herein by reference.

The present invention will now be described by way of illustration onlyby reference to the following non-limiting Sequences, Figures andExamples. Further embodiments falling within the scope of the claimswill occur to those skilled in the art in the light of these.

Sequence Listing

SEQ ID No 1 is the DNA sequence of Ad 5 with the E2 and E3 transcriptionsite mutated in accordance with the invention as shown in FIG. 2 with4xTcf inserted in place of wild type E2 promoter.

SEQ ID No 2 is the partial amino acid sequence of the 33k proteinencoded in SEQ ID No 1 unaffected by the insertion of Tcf instead of thewild type E2 promoter.

SEQ ID No 3 is the partial amino acid sequence of the pVIII proteinencoded in SEQ ID No 1 unaffected by the insertion of Tcf instead of thewild type promoter.

SEQ ID No 4 is of wild type Adenovirus VR5 in the E2/E3 region.

SEQ ID No 5 is the DNA sequence of E2 late promoter as changed in apreferred virus of the invention as shown in FIG. 4.

SEQ ID No 6 is a partial amino acid sequence 100k protein encoded in SEQID No 5 that is unaffected by the mutations in the late promoter.

SEQ ID No 7 is the DNA sequence of E1B promoter as mutated in apreferred virus of the invention as shown in FIG. 20.

SEQ ID No 8 is that of a part of the telomerase promoter site that maybe used in place of the Tcf sites exemplified herein.

SEQ ID No 9 to 25 are of primers G61 to G101 set out in the experimentalherein.

SEQ ID No 26 is that of a single Tcf binding site.

SEQ ID No 27 is that of 2 Tcf sites and flanking adenoviral DNA as foundin a preferred virus described herein.

SEQ ID No 28 is that of a mutant 3 Tcf site sequence with flanking viralDNA and an inactive Tcf site.

SEQ ID No 29 is that of a mutant 4 Tcf site sequence with flanking viralDNA as used in preferred viruses of the invention.

SEQ ID No 30 and 31 are forward and reverse primers for mutating the E1Bpromoter in a preferred virus of the invention.

SEQ ID No 32, 33 and 34 are forward and reverse primers and a proberespectively for quantitative PCR measurement of E2 expression in Taqmanprotocol.

SEQ ID No 35, 36 and 37 are forward and reverse primers and a proberespectively for quantitative PCR measurement of E3 expression in Taqmanprotocol.

SEQ ID No 38, 39 and 40 are forward and reverse primers and a proberespectively for quantitative PCR measurement of E1B expression inTaqman protocol.

SEQ ID No 41, 42 and 43 are forward and reverse primers and a proberespectively for quantitative PCR measurement of E4 expression in Taqmanprotocol.

SEQ ID No 44 and 45 are forward and reverse primers for fibre expressionmeasurement.

Sequences are also provided in the Example and figures below showingprimers and end construct virus sequences and their features ofinterest.

FIGURES

FIG. 1 shows wild type E2 region position where transcription factorsites are inserted in preferred adenoviruses of the invention (SEQ IDNO:46).

FIG. 2 shows E3 region changes to wild type virus in a preferred virus(SEQ ID NO:47).

FIG. 3 shows E2/E3 region of wild type virus (SEQ ID NO:4).

FIG. 4 shows E2 late promoter changes to wild type in a furtherpreferred virus (SEQ ID NO:5).

FIG. 5 shows a diagrammatic representation illustrating steps taken inproduction of viruses of the invention.

FIG. 6 is a diagrammatic representation of plasmid pNκBAC39.

FIG. 7 is a diagrammatic representation of plasmid p680.

FIG. 8 is a slot blot of virus infected cells probed with adenovirus DNA(A) or human genomic DNA (loading control B).

FIG. 9 is a graphic representation of results of quantatative PCR ofviral DNA from virus infected cells.

FIG. 10 is a histogram showing E2 early expression in SW 480 and H1299cell lines infected with wild type Ad5, and vMB12, vMB13 and vMB14 ofthe invention as measured by Taqman RT-PCR.

FIG. 11 is a histogram showing E3 expression in SW480 and H1299 celllines infected with wild type Ad5, and vMB12, vMB13 and vMB14 of theinvention as measured by Taqman RT-PCR.

FIG. 12 is a histogram showing DNA replication of wild type Ad5, andvMB12, vMB13 and vMB14 of the invention in SW480 and H1299 cell lines asmeasured by Taqman RT-PCR.

FIG. 13 is a histogram showing burst size (arbitrary units) with wildtype Ad5, and vMB12, vMB13 and vMB14 of the invention in SW480, H1299and W138 (fibroblast) cell lines.

FIG. 14 is a graph showing CPE results % v particles/cell of wild typeAd5, and vMB12, vMB13 and vMB14 of the invention in SW480

FIG. 15 is a graph showing CPE results % v particles/cell of wild typeAd5, and vMB12, vMB13 and vMB14 of the invention in H1299.

FIG. 16 is a plot of tumour volume v days after adminstration of buffer,vMB12, vMB13 and vMB14 in Co115 xenografts.

FIG. 17 is a histogram showing E1B-55k expression in SW480 and H1299cell lines infected with wt Ad5, vMB14 and vMB19 of the invention.

FIGS. 18, 19 and 20 are histograms of E2 and E3 early expression and DNAreplication respectively in SW480 and H1299 cells.

FIG. 21 is that of EIB promoter (SEQ ID NO:7) changes as compared to Ad5(SEQ ID NO:48) in a preferred construct or virus of the invention.

EXAMPLES

The inventors have constructed viruses with the amino-terminus of E1B55K fused to GFP (comparative virus LGM), with replacement of the E2promoter by three Tcf sites (virus Ad-Tcf3), and with the two combined(virus LGC). The inventors have also constructed viruses withreplacement of the E2 promoter by four Tcf sites alone (virus vMB12),with replacement of the E2 promoter by four Tcf sites combined withsilent mutations in the E3 promoter, particularly to NF1, NFκB, AP1, andATF sites (virus vMB14), and with replacement of the E2 promoter by fourTcf sites combined with silent mutations in the E3 promoter,particularly to NF1, NFκB, AP1, but not ATF sites (virus vMB13). Theinventors have also constructed viruses with replacement of the Sp1 sitein the E1B promoter with four Tcf sites in a wild type adenovirusbackbone (virus vMB23), in a vMB12 backbone (virus vMB27), in a vMB13backbone (virus vMB31) and in a vMB14 backbone (virus vMB19).

Brief Description of Key Constructs:

Gap repair and two step gene replacement as used to construct the AdTcf3 YAC/BAC (pMB36) are illustrated in FIG. 5 (all of the steps showntake place inside a yeast cell). Maps of pNKBAC39 and p680 are shown inFIGS. 6 and 7. Availability of materials: pUC19 (Clontech), Bluescript(Stratagene), pEGFP-C1 (Clontech), phGFP-S65T (Clontech) and pRS406(Stratagene).

YAC/BAC (pMB19):

The adenovirus genome was modified in a large plasmid with a bacterialartificial chromosome (F′) replication origin, a yeast centromere andreplication origin, and selectable markers for yeast and bacteria (HIS3,chloramphenicol resistance gene).

Ad5 YAC/BAC (pMB20):

Genomic DNA was prepared from adenovirus type 5 obtained from ATCC(VR5). Small terminal fragments were amplified by PCR and cloned intothe YAC/BAC. The vector was linearised at a site between the twoterminal Ad5 fragments and transfected into yeast together with fulllength Ad5 genomic DNA. The plasmid was recircularised by homologousrecombination (gap repair), giving full length Ad5 genomic DNA cloned inthe YAC/BAC.

E1B::GFP fusion (pMB25): An Ad5 fragment containing the part of E1B 55kwhich overlaps E1B 19k was cloned by PCR and fused to the 5′-end ofEGFP. This was then embedded in a larger Ad5 fragment, so that theE1B::GFP fusion was flanked on both sides by Ad5 sequence, in a vectorcontaining LEU2 and CYH2 for selection and counter-selection in yeast.

LGM YAC/BAC (pMB26):

The E1B::GFP fusion in pMB25 was inserted in the Ad5 YAC/BAC by two stepgene replacement. The resulting plasmid was transferred to E. coli toallow production of enough DNA for sequencing and transfection intomammalian cells. Plasmid from E. coli was cut with PacI to liberate theAd 5 insert, then transfected into 293 cells to make virus.

E2-Tcfx3/4 Mutations (pMB33/69):

An Ad5 fragment containing the E2 region was cloned into a vectorcontaining URA3 for selection and counterselection in yeast. The E2promoter was replaced by inverse PCR. pMB33 contains 3 Tcf sites; pMB69contains 4 Tcf sites.

Ad-Tcfx3 YAC/BAC (pMB36):

The E2-Tcfx3 replacement sequence in pMB33 was inserted in the Ad5YAC/BAC by two step gene replacement. The YAC/BAC was then transferredto E. coli and 293 cells to make virus. The resulting virus is calledAd-Tcf3.

Ad-Tcfx4 YAC/BAC (pMB74):

The E2-Tcfx4 replacement sequence in pMB69 was inserted in the Ad5YAC/BAC by two step gene replacement. The YAC/BAC was then transferredto E. coli and 293 cells to make virus. The resulting virus is calledvMB12.

LGC YAC/BAC (pMB37):

The E2-Tcfx3 mutations in pMB33 were inserted in the LGM YAC/BAC by twostep gene replacement. The YAC/BAC was then transferred to E. coli and293 cells to make virus.

E2-Tcfx4/E3 Mutations (pMB66):

The E2 and E3 mutations were introduced in three successive rounds ofinverse PCR. The E2 changes were made as for pMB33 but with four Tcfsites. To permit two step gene replacement some additional Ad 5 sequencewas added 3′ of the E3 promoter.

Ad-Tcfx4/mutE3 YAC/BAC (pMB75):

The E3 mutations and E2-Tcfx4 replacement sequence in pMB66 wereinserted in the Ad5 YAC/BAC by two step gene replacement. The YAC/BACwas then transferred to E. coli and 293 cells to make virus. Theresulting virus is called vMB14.

Ad-Tcfx4/mutE3+ATF YAC/BAC (pMB73):

The E3 mutations, except the ATF mutations, and E2-Tcfx4 replacementsequence in pMB66 were inserted in the Ad5 YAC/BAC by two step genereplacement. The YAC/BAC was then transferred to E. coli and 293 cellsto make virus. The resulting virus is called vMB13.

Note: Both pMB73 and 75 were made by two step gene replacement in theAd5 YAC/BAC using the pMB66. Mutations near the site of integration arenot always transferred by two step gene replacement. pMB73 lacks the ATFsite mutations because the ATF site is the nearest to the site ofintegration.

Detailed Procedures:

pMB20:

Ad5 genomic DNA was gap repaired into pMB19 cut with SalI. pMB19 wasmade by inserting a yeast replication origin (from pH4ARS, Bouton andSmith, 1986) into the SacI site of a vector already containing theterminal Ad5 fragments (pMB 10). The starting vector (pNKBAC39, Larionovet al., 1996) was expected not to need an ARS but this assumption provedincorrect. The Ad5 terminal fragments were cloned initially by PCR intoa pUC19-derived vector (to give pMB1 and pMB2), and then transferredsequentially into the BamHI/Bsu36I sites of pNKBAC39. PacI sites werepresent in the G76 primer used to make pMB1 and pMB2 (PCR with primersG74-G76 and G75-G76 giving Ad5 fragments of 390 and 356 bp).

pMB25:

The EGFP vector is a modified vector from Clontech. It has the 5′ end ofGFP from pEGFP-C1 and the 3′-end of GFP from phGFP-S65T. The Ad5 PCRfragment (nt 2019-2261, primers G77-78) was cloned into the NheI andAgeI sites at the 5′-end of EGFP to give pMB7. The SmaI Ad5 fragment (nt1007-3940) containing the E1 region was cloned into Bluescript to givepMB22. The E1B::GFP fusion (NotI/KpnI) was cloned into pMB22(BglII/KpnI) to give pMB24. The XhoI/BamHI fragment of pMB24 containingthe Ad5 insert was cloned into p680 (Ketner et al., 1994) to give pMB25.

pMB33:

The Ad5 PCR fragment (nt 26688-27593, PCR with primers G61-G62, productcut with SacI/KpnI) was cloned into the KpnI/SacI sites in pRS406 togive pMB32. This was mutagenised by inverse PCR to insert the Tcf sitesusing primers G63-G64. The primers should give four Tcf sites but thefirst Tcf site was subsequently found to contain a mutation, so thefinal vector only contains three Tcf sites.

pMB66:

pMB33 with four correct Tcf sites is called pMB69. pMB69 was mutagenisedby inverse PCR using primers G89 and G90 to give pMB46. pMB46 wasmutagenised by inverse PCR using primers G87 and G88 to give pMB49,which contains four Tcf sites in E2 and all of the desired mutations inE3. The Ad 5 PCR fragment (nt 27331-27689, primers G100-G101) used tofacilitate two step gene replacement was cut with EcoRI and PstI andcloned into Bluescript to give pMB58. This 3′ extension was first usedto create a vector with four Tcf sites in E2 and a wild type E3 promoter(the EcoRI/PstI fragment from pMB58 was inserted into the EcoRI/SacIsites in pMB49) to give pMB63. The E3 mutations were then cloned backinto this vector (the SacI/KpnI fragment of pMB49 was cloned into theSacI/KpnI sites in pMB63) to give pMB66.

pMB67:

The E2 promoter with two Tcf sites was constructed as for pMB33 butusing primers G91 and G92 for the inverse PCR, to give pMB45. AnEcoRI/Eco47III fragment containing the two Tcf sites was transferredfrom pMB45 into pMB66 to give pMB67.

Primers G61   Ad 5,26688 (E2 region) (SEQ ID NO:9)5′-TGCATTGGTACCGTCATCTCTA-3′ G62   Ad 5, 27882 (E2 region) (SEQ IDNO:10) 5′-GTTGCTCTGCCTCTCCACTT-3′ G63   iPCR, E2 promoter replacement (2x Tcf), upper primer (SEQ ID NO:11)5′-CAGATCAAAGGGATTAAGATCAAAGGGCCATTATGAGCAAG-3′ G64   iPCR, E2 promoterreplacement (2 x Tcf), lower primer (SEQ ID NO:12)5′-GATCCCTTTGATCTCCAACCCTTTGATCTAGTCCTTAAGAGTC-3′ G74   Ad5, 390 (leftarm gap repair fragment) (SEQ ID NO:13) 5′-GGG GGA GTC TCC ACG TAAACG-3′ G75   Ad5, 36581 (right arm gap repair fragment) (SEQ ID NO:14)5′-GGG CAC CAG CTC AAT CAG TCA-3′ G76   Ad5 ITR plus EcoRI, HindiIII andPacI sites (SEQ ID NO:15) 5′-CGG AAT TCA AGC TTA ATT AAC ATC ATC AAT AATATA CC-3′ G77   Ad 5, 2020 (E1B fragment plus NheI site) (SEQ ID NO:16)5′-GCG GCT AGC CAC CAT GGA GCG AAG AAA CCC A-3′ G78   Ad 5, 2261 (E1Bfragment plus AgeI site) (SEQ ID NO:17) 5′-GCC ACC GGT ACA ACA TTCATT-3′ G87   iPCR to destroy the E3 NF-1, Li and L2 binding sites, upperprimer (SEQ ID NO:18) 5′-AGCTGGGCTCTCTTGGTACACCAGTGCAGCGGGCCAACTA-3′G88   iPCR to destroy the E3 NF-1, Li and L2 binding sites, lower primer(SEQ ID NO:19) 5′-CCCACCACTGTAGTGCTGCCAAGAGACGCCCAGGCCGAAGTT-3′G89   iPCR to destroy the E3 ATF and AP-1 binding sites, upper primer(SEQ ID NO:20) 5′-CTGCGCCCCGCTATTGGTCATCTGAACTTCGGCCTG-3′ G90   iPCR todestroy the E3 ATF and AP-1 binding sites, lower primer (SEQ ID NO:21)5′-CTGCGGGCGGCTTTAGACACAGGGTGCGGTC-3 ′ G91   iPCR, E2 promoterreplacement (1 x Tcf), upper primer (SEQ ID NO:22)5′-CAGATCAAAGGGCCATTATGAGCAAG-3′ G92   iPCR, E2 promoter replacement (1x Tcf), lower primer (SEQ ID NO:23) 5′-GATCCCTTTGATCTAGTCCTTAAGAGTC-3′G100  Ad 5, 27757 (E3 distal promoter region) (SEQ ID NO:24)5′-ATGGCACAAACTCCTCAATAA-3′ G101  Ad 5, 27245 (E3 distal promoterregion) (SEQ ID NO:25) 5′-CCAAGACTACTCAACCCGAATA-3′

The following references for procedures are incorporated herein byreference:

Bouton, A. H., and Smith, M. M. (1986). Fine-structure analysis of theDNA sequence requirements for autonomous replication of Saccharomycescerevisiae plasmids. Mol Cell Biol 6, 2354-63.

Ketner, G., Spencer, F., Tugendreich, S., Connelly, C., and Hieter, P.(1994). Efficient manipulation of the human adenovirus genome as aninfectious yeast artificial chromosome clone. Proc Natl Acad Sci U S A91, 6186-90.

Larionov, V., Kouprina, N., Graves, J., Chen, X. N., Korenberg, J. R.,and Resnick, M. A. (1996). Specific cloning of human DNA as yeastartificial chromosomes by transformation-associated recombination. ProcNatl Acad Sci U S A 93, 491-6.

E2 Promoter Replacement Sequences Inserts for Preparing Ad-Tcf Viruses

single Tcf site: (SEQ ID NO:26) AGATCAAAGGG Ad 5 sequence: (SEQ IDNO:27) GACTAG-...-GCCATT 2 Tcf sites: (SEQ ID NO:28)GACTAG-ATCAAAGGGATCCAGATCAAAGG-GCCATT 3 Tcf sites: (SEQ ID NO:29)GACTAG-ATCAAGGGTTGGAGATCAAAGGGATCCAGATCAAAGGGATTAA GAT CAAAGG-GCCATT 4Tcf sites: (SEQ ID NO:30)GACTAG-ATCAAAGGGTTGGAGATCAAAGGGATCCAGATCAAAGGGATTA AGATCAAAGG-GCCATT

Note: The 3 Tcf vector is a mutant form of the 4 Tcf vector resultingfrom a PCR cloning artefact (there is a single A deletion in the firstTcf site). This is the sequence present in the Ad-Tcf3 and LGC viruses.

E3 Promoter Binding Sites

Four sites have been identified in the E3 promoter by DNase Ifoot-printing in Hela cells (Garcia 1987, Hurst 1987) Site 1 covers theTATA box, the remaining sites (underlined and marked H2-H4 in FIGS. 1 to3) are bound by ATF, AP1 and NF1. Two sites have been identified byDNase I footprinting in lymphoid cells (Williams 1990) (underlined andmarked L1 and L2 in FIGS. 1 to 3), they bind NFκB family members.

To inactivate the promoter, mutations were introduced by inverse PCR.All sites except the TATA box contain at least one mutation. Themutations are silent at the protein level. The L1, L2 and H2 boxescontain multiple substitutions of highly conserved residues. The H4 boxcontains a single mutation in a relatively poorly conserved residuebecause of the limited choice of alternative codons. Mutation of the H4site was not a priority because deletion of this site has no effect onE3 transcription in Hela cells (Garcia 1987). Only relatively weaklyconserved residues in the H3 box were mutated because any modificationof the most conserved residues in the published AP1 site (TGAC) wouldchange the protein sequence. There is a better match to an AP1 siteslightly 3′ of the published site (still within the publishedfootprint); the mutations introduced completely destroy this site.

Construction of Viruses Containing 4 Tcf Sites Controlling the E1BPromoter.

pMB22 (E1 region in Bluescript) is described in the construction of theLGM virus.

1. pRDI-238=Introduction of Tcf sites and deletion of Sp1 site byinverse PCR from pMB22. Primers:tCCCTTTGATCTccaaCCCTTTGATCTAGTCCtatataatgcgccgtg (SEQ ID NO:30) andtccAGATCAAAGGGattaAGATCAAAGGGatttaacacgccatgcaa (SEQ ID NO:31). Theunderlined sequence between the Tcf site and TATA box is identical withthat in the E2 promoter (ie not E1B promoter sequence) because we knowthat this spacing and sequence are compatible with good regulation ofthe promoter.

2. pRDI-239=Transfer small fragment containing the Tcf mutations into ayeast integrating vector. The pRDI-238 EcoRI/SacI fragment was clonedinto the same sites in pRS406.

3. pRDI-241=E1B-Tcf yeast integrating vector. The E1B-containing 2 kbSacI fragment from pMB22 was cloned into the same site in pRDI-239 toprovide additional sequences for recombination with the YAC/BAC.

4. Two step gene replacement in YAC/BACs. pRDI-241 was cut with XbaI andtransfected into yeast (yMB strains) containing the adeno YAC/BACs. Toselect for integration and excision, the transformants were plated onura- then 5-FOA medium. Plasmids were then rescued to DH10B:

pRDI-243=recombinant from yMB2 (wild type Ad5)

pRDI-268=recombinant from yMB15 (4xTcf-E2, wt E3)

pRDI-254=recombinant from yMB13 (4xTcf-E2, mutant E3+wt ATF)

pRDI-264=recombinant from yMB17 (4xTcf-E2, fully mutant E3)

5. These plasmids were cut with PacI and transfected into 293 cells(ATCC CRL 1573) containing activated Tcf (ΔNβ-catenin, supplied by Dr HClevers) to produce recombinant adenoviruses:

vMB15=virus pool derived from pRDI-243 transfection

vMB16=virus pool derived from pRDI-268 transfection

vMB17=virus pool derived from pRDI-254 transfection

vMB18=virus pool derived from pRDI-264 transfection

6. The viruses were plaque purified on SW480 cells because these cellscontain active Tcf and have no endogenous E1B sequences with which theviral genome could recombine. The viruses were then expanded on SW480,purified by Cs banding, and checked by restriction digestion andsequencing in the E1B and E2/E3 regions. The plaque purified viruseswere given the following names:

vMB23=Ad5 with 4 x Tcf sites in the E1B promoter

vMB27=Ad5 with 4 x Tcf sites in both the E1B and E2 promoters

vMB31=Ad5 with 4 x Tcf sites in the E1B and E2 promoters, and a mutantE3 promoter with a wild type ATF site

vMB19=Ad5 with 4 x Tcf sites in the E1B and E2 promoters, and a fullymutant E3 promoter

Results:

Luciferase assays using Tcf reporters show that p53-mutant lungcarcinoma cells (H1299) lack Tcf activity and p53-mutant colon carcinomacells (SW480) have strong Tcf activity. Viruses selective for cellscontaining Tcf activity should therefore replicate in SW480 but notH1299. The inventors have demonstrated that matched viruses withTcf-mutant E2 promoters express E2 gene products preferentially bywestern blotting; replicate better by slot blotting and quantitativePCR; and have greater cytopathic effect in SW480 than H1299. Therelatively modest effect of an E2 promoter mutation alone isconsiderably enhanced when the virus also lacks E1B 55k. E1B 55kmutations reduce nuclear export of DBP mRNA transcribed from the E2 latepromoter. DBP can be expressed from both the early and late E2promoters. The inventors have determined that the ElB 55k-dependentreduction in DBP expression might be rescued by the Tcf mutations in theE2 early promoter. Consistent with this the level of DBP expressed fromLGC is significantly reduced in H1299. This effect of E1B 55k isentirely independent of p53, as is obvious from the fact that neithercell line contains p53.

Western Blot

H1299 and SW480 were infected at an moi of 0.2 with wild type Ad5,Ad-Tcf3, LGM and LGC. Cells were harvested for western blotting after24, 48 and 72 hours. Blots were probed with antibodies against DBP. Wildtype Ad5 gave comparable expression of protein in SW480 and H1299.Ad-Tcf3 gave slightly stronger expression of DBP in SW480 than in H1299.Ad-Tcf3 gave higher expression of DBP protein than wild type Ad5 inSW480 at 24 hours, but was similar to wild type Ad5 at 48 and 72 hours.DBP was expressed better by LGM than LGC in H1299, but both viruses gavecomparable DBP expression in SW480.

The results are consistent with the Tcf-mutant E2 promoter being morestrongly activated in colon cells than in lung cells.

Viral DNA Replication Assays

H1299 and SW480 were infected at an moi of 0.2 with wild type Ad5,Ad-Tcf3, LGM and LGC. Cells were harvested for DNA extraction after 0,24, 48 and 72 hours. All of the samples were slot blotted and hybridisedto ³²P-labelled adenoviral DNA (FIG. 8A) or control human genomic DNA(FIG. 8B). This showed replication of LGM in both cell lines, butreplication of LGC only in SW480 (the actual values measured byphosphorimager are equal to background). To calculate the differencemore precisely, the 72 hour samples were tested by Taqman quantitativePCR assay (Perkin Elmer) using Ad5 primers (FIG. 9). This confirmed theresults of the slot blot: LGC is two-fold worse than wild type Ad5 inSW480, but 3000-fold worse in H1299. LGM is ten-fold worse than wildtype Ad5 in SW480, but four-fold worse in H1299. Ad-Tcf3 also shows someselectivity for SW480 cells, albeit less dramatic: it is 1.3-fold betterthan wild type Ad5 in SW480, but 7.4-fold worse in H1299.

These results are consistent with the stronger expression of pol and pTPin Tcf-mutant E2 promoter viruses in colon cells resulting in greaterviral replication. Additionally, the data show that the combination ofan E2 promoter mutation with deletion of E1B 55k results inexceptionally little replication in cells lacking Tcf activity.

Cytopathic Effect (CPE) Assays

H1299 and SW480 were infected with five-fold dilutions of wild type AdS,Ad-Tcf3, LGM and LGC (moi of 0.6 in well 1, moi of 0.001 in well 5).Dishes were stained with crystal violet after eight days. CPE isapparent with wild type Ad5 in H1299 even at the highest dilution. Wildtype Ad5 is five-fold less active in SW480 than H1299.

Ad-Tcf3 is five-fold less active than wild type Ad5 in H1299. LGM is125-fold less active than wild type Ad5 in H1299. LGC is 625-fold lessactive than wild type AdS in H1299. The Tcf3 mutation in the E2 promoterthus results in a five-fold reduction in CPE in H1299. Ad-Tcf3 issimilar to wild type Ad5 in SW480, indicating that there is a five-foldgain in activity in cells containing Tcf activity. LGC is five-fold moreactive than LGM in SW480.

LGC is 625-fold less active than wild type Ad5 in cells lacking Tcfactivity, but only five-fold less active in cells containing Tcfactivity. This represents a 125-fold selectivity for colon cells, and afive-fold greater activity than a simple E1B 55k-deficient virus.

Additional CPE assays were performed with SW480 cells and normal lungfibroblasts using the above viruses Ad-Tcf3, LGM and LGC. Ad-Tcf3 gavewild type activity on SW480 but 5-fold less activity on normalfibroblasts. In this experiment LGM and LGC both gave about 125-foldless activity than wild type Ad5 on SW480 but 5-fold and >125-fold lessactivity respectively on normal fibroblasts.

The selectivity and efficacy of Ad-Tcf 3 are shown to be greater thanthat of LGM which itself has essentially the same properties as theONYX-015 virus.

4xTcf Viruses vMB12, 13, 14 and 19

Methods:

Cell lines: SW480 and Co115 colorectal carcinoma cells were supplied byDr B Sordat. W138 cells were supplied by ATCC. The H1299 cells weresupplied by Dr C Prives and contain an integrated tet-VP16transactivator (Chen, X., et al. 1996. Genes Dev 10, 2438-51).

Taqman Assays

Cells were infected with either 300 (SW480 and H1299) or 1000 (WI38)viral particles/cell in DMEM 10% FCS. Two hours after infection, themedium was removed and replaced with 2 ml of fresh DMEM 10% FCScontaining 10 mM hydroxyurea. Twenty-four hours after infection, cellsare washed with 1× PBS and lysed with either buffer RLT (from QiagenRNeasy Mini Kit, Ref. 74104) for RNA extraction or with a mix containing1× PBS, buffer AL and proteinase K solution (from Qiagen DNeasy TissueKit, Ref. 69504) for DNA extraction. RNA and DNA extractions wereperformed according to the manufacturer's instructions. Reversetranscription (RT) was performed using 1 μg of total RNA and MMLVSuperscript Core Reagents (LifeTechnologies, Ref. 18064022) in 20 μlreaction volume. TaqMan PCR reactions were performed using TaqManUniversal PCR Master Mix Kit (Perkin Elmer, Ref. 4304447), 900 nM ofprimers (Microsynth and Eurogentec) and 500 nM of TaqMan probe(Eurogentec). Sybr green PCR reactions were performed using Sybr greenUniversal PCR Master Mix Kit (Perkin Elmer, Ref. 4309155) and 900 nM offibre gene primers (Eurogentec). The amount of template used for the PCRreaction was 1 μg of genomic DNA or to 5 μl of RT reaction volume.Results for DNA were normalised to OD₂₆₀, results for RNA werenormalised to ribosomal RNA (Ribosomal RNA Control, Perkin Elmer, ref4310893E). The primers and probes for quantitative PCR were

E2 early forward primer: TTCGCTTTTGTGATACAGGCA (SEQ ID NO:32) E2 earlyreverse primer: GTCTTGGACGCGACGAGAAG (SEQ ID NO:33) E2 probe:CGGAGCGTTTGCCGCGC (SEQ ID NO:34) E3 forward primer:AGCTCGGAGAGGTTCTCTCGTAG (SEQ ID NO:35) E3 reverse primer:AACACCTGGTCCACTGTCGC (SEQ ID NO:36) E3 probe: CCGCGACTCCGTTTCAACCCAGA(SEQ ID NO:37) E1B-55k forward primer: TGCTTCCATCAAACGAGTTGG (SEQ IDNO:38) E1B-55k reverse primer: GCGCTGAGTTTGGCTCTAGC (SEQ ID NO:39) ElB-55k probe: CGGCGGCTGCTCAATCTGTATCTTCA (SEQ ID NO:40) E4 forwardprimer: GGTTGATTCATCGGTCAGTGC (SEQ ID NO:41) E4 reverse primer:ACGCCTGCGGGTATGTATTC (SEQ ID NO:42) E4 probe: AAAAGCGACCGAAATAGCCCG (SEQID NO:43) Fibre forward primer: TGATGTTTGACGCTACAGCCATA (SEQ ID NO:44)Fibre reverse primer: GGGATTTGTGTTTGGTGCATTAG (SEQ ID NO:45)

Western Blot Analysis

Cells were infected with either 300 (SW480 and H1299) or 1000 (WI38)viral particles/cell in DMEM 10% FCS. Two hours after infection, themedium was removed and replaced with 2 ml of fresh DMEM 10% FCS. Cellswere harvested 24 hours after infection. Immunoblotting was performed asdescribed by Harlow and Lane (Antibodies: A laboratory manual, New York:Cold Spring Harbor Laboratory Press, 1988 incorporated herein byreference). Dr. A. Levine provided both 2A6 anti-E1B55K antibody (SarnowP. et al, Virology, 1982, 120: 510-517) and B6 anti-DBP antibody (ReichN. et al, Virology, 1983, 128: 480-484). Detection was withHRP-conjugated rabbit anti-mouse IgG (DAKO A/S, Denmark) andchemiluminescence (Amersham, Little Chalfont, UK).

Plaque Assay

Cells were infected in duplicate with either 300 (for SW480 and H1299)or 1000 (WI38) viral particles/cell in DMEM 10% FCS. After two hoursincubation at 37° C., the medium was removed and replaced with 2 ml offresh DMEM 10% FCS. After an additional 48 hours, the cells were scrapedinto the culture medium and lysed by three cycles of freeze-thawing. Thesupernatant of each duplicate point was tested for virus production bytriplicate plaque assay for 8-10 days under semisolid agarose on SW480cells

Cytopathic Effect Assays

For the cytopathic effect assays, SW480 and H1299 cells were plated intriplicate in 96-well plates and infected with five-fold dilutions ofvirus from 1000 to 0.06 viral particles/cell. After two hours incubationat 37° C., an additional 100 μl of DMEM 10% FCS was added to each well.After six days, the assays was terminated and the protein content ineach well was measured using the BCA Protein Assay Kit (Pierce,Rockford, Ill., USA) according to the manufacturer's instructions.

Xenografts

One million Co115 cells were injected subcutaneously into the flank ofnude mice. On days 4-8 after injection, 3×10¹⁰ particles of virus orbuffer were injected directly into the tumour. Tumour size was measuredin two dimensions with callipers and tumour volume was calculated usingthe formula: volume=0.4×length×width².

Cotton Rat Lungs

Cotton rats were infected intranasally with 3×10¹⁰ particles of virus in50 μl of buffer. Three days later the animals were killed withisoflurane and four fragments of lung (from left and right upper andlower lobes) were taken from each animal. DNA was extracted by QiagenDNeasy Tissue Kit and the samples from each animal pooled. Viral DNAcontent was determined by Taqman PCR with E4 primers and probe using 100ng input DNA, as described above.

Results: vMB12, 13, 14 and 19

The viruses with four Tcf sites in the E2 promoter were tested in SW480,H1299 and W138 cells. Cells were harvested 24 hours after infection.Western blotting for DBP showed that DBP is expressed at least as wellby the mutant viruses as by wild type in SW480, but much worse in H1299and W138 cells:

Taqman RT-PCR transcription assays (see FIG. 10) show mutant viruses tohave wild type levels of E2 mRNA in SW480 cells, but reduced levels inH1299 cells:

The Taqman assay further demonstrates that mutation of the E3 promoterin vMB14 decreases both E2 (FIG. 10) and E3 mRNA levels (FIG. 11):

To determine whether DNA replication is affected by the promotermutations, SW480 and H1299 cells were infected with wild type, vMB12, 13and 14, and harvested at 24 hours. Sybr green PCR assays using primersfrom the fibre region show that DNA replication is normal in SW480 butreduced in H1299 cells (FIG. 12):

To determine whether virus replication is affected by the promotermutations, SW480 and H1299 cells were infected with wild type, vMB12, 13and 14, and harvested at 48 hours. Cells were lysed by freeze-thawingand virus production was measured by plaque forming assay on SW480cells. This showed that the mutant viruses are comparable or better thanwild type in SW480 cells, but defective in H1299 and W138 cells (SeeFIG. 13):

To determine whether the viruses show selective toxicity to colon cells,cytopathic effect assays were performed on SW480 and H1299 cells. Thisshowed that the mutant viruses are comparable to wild type in SW480cells (FIG. 14) but showed reduced cytopathic effect in H1299 cells(FIG. 15):

To determine whether the viruses show a therapeutic effect in vivo, theywere injected into Co115 colon carcinoma xenografts in nude mice. Thisshowed that intratumoral injection of all of the viruses delay thegrowth of xenografted colon tumours and prolong the survival of nudemice. vMB12 and 13 were more effective than vMB14 (FIG. 16):

E1B expression is required for induction of inflammatory damage byadenoviruses in cotton rat lung (Ginsberg, H. S., et al 1999. PNAS 96,10409-11). To reduce the risk of inflammatory reactions, Tcf sites werecloned into the E1B promoter. The resulting viruses should express E1Bgene products in colon tumour cells but not in normal cells. In additionto reducing the risk of inflammatory reactions, this could also reduceexpression of E2 gene products from the E2 late mRNA, because E1B 55k isreported to be required for E2 late mRNA export. The virus with themutant E1B promoter cloned into the vMB14 backbone is called vMB19. Wildtype, vMB14 and vMB19 were tested in SW480 and H1299 cells. Cells wereharvested 24 hours after infection. Western blotting for E1B and DBPshowed that E1B 55k is expressed in SW480 but not in H1299. vMB14 and 19gave similar DBP expression in H1299 cells, suggesting that there is nota large effect of E1B on DBP late mRNA export, at least at 24 hours inthis cell line:

Taqman RT-PCR transcription assays 24 hours after infection confirmedthat the vMB19 has wild type levels of E1B, E2 and E3 mRNA in SW480cells, but reduced levels in H1299 cells (FIGS. 17, 18 and 19):

Viral DNA replication was tested 24 hours after infection by Sybr greenquantitative PCR using fibre primers. This showed that both mutantviruses replicate normally in SW480 cells but are defective in H1299cells. Both mutant viruses were comparable, again suggesting that thedocumented reduction in E1B 55k expression does not have a marked effectin this experiment (FIG. 20):

To determine whether vMB19 shows reduced DNA replication in normal cellsin an in vivo setting, cotton rats were infected intra-nasally withvirus and DNA was extracted from lungs three days later. Rats weretreated in groups of five for each virus. Taqman quantitative PCR usingE4 primers showed a median 50-fold reduction in viral DNA concentrationwith vMB19 compared to wild type.

Conclusions: vMB12, 13, 14 and 19

These data show that

1. insertion of Tcf sites in the E2 promoter is compatible with theproduction of viable adenovirus

2. the mutant E2 promoter is more active in colon tumour cells thannon-colon tumour cells

3. mutation of the E3 promoter further reduces E2 activity in non-colontumour cells

4. these mutations reduce viral DNA replication, virus replication andcytopathic effect in non-colon tumour cells

5. the mutant viruses impair the growth of xenografts in nude mice

6. insertion of Tcf sites in the E1B promoter is compatible with theproduction of viable adenovirus

7. the mutant E1B promoter is more active in colon tumour cells thannon-colon tumour cells

8. in the models tested so far the E1B promoter mutation does not affectvirus replication

9. the virus with combined E1B and E2/E3 promoter mutations showsreduced replication in cotton rat lungs, and is expected to produce lessinflammatory damage than viruses with the E2/E3 mutations alone.

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                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 45 <210> SEQ ID NO 1 <211> LENGTH: 752<212> TYPE: DNA <213> ORGANISM: ADENOVIRUS VR5 <220> FEATURE:<221> NAME/KEY: promoter <222> LOCATION: (250)..(302) <400> SEQUENCE: 1ggagcgctgc gtctggcgcc caacgaaccc gtatcgaccc gcgagcttag aa#acaggatt     60tttcccactc tgtatgctat atttcaacag agcaggggcc aagaacaaga gc#tgaaaata    120aaaaacaggt ctctgcgatc cctcacccgc agctgcctgt atcacaaaag cg#aagatcag    180cttcggcgca cgctggaaga cgcggaggct ctcttcagta aatactgcgc gc#tgactctt    240aaggactaga tcaaagggtt ggagatcaaa gggatccaga tcaaagggat ta#agatcaaa    300gggccattat gagcaaggaa attcccacgc cctacatgtg gagttaccag cc#acaaatgg    360gacttgcggc tggagctgcc caagactact caacccgaat aaactacatg ag#cgcgggac    420cccacatgat atcccgggtc aacggaatcc gcgcccaccg aaaccgaatt ct#cttggaac    480aggcggctat taccaccaca cctcgtaata accttaatcc ccgtagttgg cc#cgctgcac    540tggtgtacca agagagccca gctcccacca ctgtagtgct gccaagagac gc#ccaggccg    600aagttcagat gaccaatagc ggggcgcagc ttgcgggcgg ctttagacac ag#ggtgcggt    660cgcccgggca gggtataact cacctgacaa tcagagggcg aggtattcag ct#caacgacg    720 agtcggtgag ctcctcgctt ggtctccgtc cg       #                   #         752 <210> SEQ ID NO 2 <211> LENGTH: 82<212> TYPE: PRT <213> ORGANISM: ADENOVIRUS VR5 <400> SEQUENCE: 2Gly Ala Leu Arg Leu Ala Pro Asn Glu Pro Va #l Ser Thr Arg Glu Leu  1               5  #                 10  #                 15Arg Asn Arg Ile Phe Pro Thr Leu Tyr Ala Il #e Phe Gln Gln Ser Arg             20      #             25      #             30Gly Gln Glu Gln Glu Leu Lys Ile Lys Asn Ar #g Ser Leu Arg Ser Leu         35          #         40          #         45Thr Arg Ser Cys Leu Tyr His Lys Ser Glu As #p Gln Leu Arg Arg Thr     50              #     55              #     60Leu Glu Asp Ala Glu Ala Leu Phe Ser Lys Ty #r Cys Ala Leu Thr Leu 65                  # 70                  # 75                  # 80Lys Asp <210> SEQ ID NO 3 <211> LENGTH: 148 <212> TYPE: PRT<213> ORGANISM: ADENOVIRUS VR5 <400> SEQUENCE: 3Met Ser Lys Glu Ile Pro Thr Pro Tyr Met Tr #p Ser Tyr Gln Pro Gln  1               5  #                 10  #                 15Met Gly Leu Ala Ala Gly Ala Ala Gln Asp Ty #r Ser Thr Arg Ile Asn             20      #             25      #             30Tyr Met Ser Ala Gly Pro His Met Ile Ser Ar #g Val Asn Gly Ile Arg         35          #         40          #         45Ala His Arg Asn Arg Ile Leu Leu Glu Gln Al #a Ala Ile Thr Thr Thr     50              #     55              #     60Pro Arg Asn Asn Leu Asn Pro Arg Ser Trp Pr #o Ala Ala Leu Val Tyr 65                  # 70                  # 75                  # 80Gln Glu Ser Pro Ala Pro Thr Thr Val Val Le #u Pro Arg Asp Ala Gln                 85  #                 90  #                 95Ala Glu Val Gln Met Thr Asn Ser Gly Ala Gl #n Leu Ala Gly Gly Phe            100       #           105       #           110Arg His Arg Val Arg Ser Pro Gly Gln Gly Il #e Thr His Leu Thr Ile        115           #       120           #       125Arg Gly Arg Gly Ile Gln Leu Asn Asp Glu Se #r Val Ser Ser Ser Leu    130               #   135               #   140 Gly Leu Arg Pro 145<210> SEQ ID NO 4 <211> LENGTH: 780 <212> TYPE: DNA<213> ORGANISM: ADENOVIRUS VR5 <400> SEQUENCE: 4ggagcgctgc gtctggcgcc caacgaaccc gtatcgaccc gcgagcttag aa#acaggatt     60tttcccactc tgtatgctat atttcaacag agcaggggcc aagaacaaga gc#tgaaaata    120aaaaacaggt ctctgcgatc cctcacccgc agctgcctgt atcacaaaag cg#aagatcag    180cttcggcgca cgctggaaga cgcggaggct ctcttcagta aatactgcgc gc#tgactctt    240aaggactagt ttcgcgccct ttctcaaatt taagcgcgaa aactacgtca tc#tccagcgg    300ccacacccgg cgccagcacc tgtcgtcagc gccattatga gcaaggaaat tc#ccacgccc    360tacatgtgga gttaccagcc acaaatggga cttgcggctg gagctgccca ag#actactca    420acccgaataa actacatgag cgcgggaccc cacatgatat cccgggtcaa cg#gaatccgc    480gcccaccgaa accgaattct cttggaacag gcggctatta ccaccacacc tc#gtaataac    540cttaatcccc gtagttggcc cgctgccctg gtgtaccagg aaagtcccgc tc#ccaccact    600gtggtacttc ccagagacgc ccaggccgaa gttcagatga ctaactcagg gg#cgcagctt    660gcgggcggct ttcgtcacag ggtgcggtcg cccgggcagg gtataactca cc#tgacaatc    720agagggcgag gtattcagct caacgacgag tcggtgagct cctcgcttgg tc#tccgtccg    780 <210> SEQ ID NO 5 <211> LENGTH: 209 <212> TYPE: DNA<213> ORGANISM: ADENOVIRUS VR5 <400> SEQUENCE: 5tcgcctgacg aaaagtccgc ggctccgggg ttgaaactca ctccggggct gt#ggacgtcg     60gcttaccttc gcaaatttgt acctgaggac taccacgccc acgagattag gt#tctacgaa    120gatcagtccc gcccgccaaa tgcggagctt accgcctgcg tcattaccca gg#gccacatt    180 cttggccaat tgcaagccat caacaaagc         #                   #           209 <210> SEQ ID NO 6 <211> LENGTH: 69<212> TYPE: PRT <213> ORGANISM: ADENOVIRUS VR5 <400> SEQUENCE: 6Ser Pro Asp Glu Lys Ser Ala Ala Pro Gly Le #u Lys Leu Thr Pro Gly  1               5  #                 10  #                 15Leu Trp Thr Ser Ala Tyr Leu Arg Lys Phe Va #l Pro Glu Asp Tyr His             20      #             25      #             30Ala His Glu Ile Arg Phe Tyr Glu Asp Gln Se #r Arg Pro Pro Asn Ala         35          #         40          #         45Glu Leu Thr Ala Cys Val Ile Thr Gln Gly Hi #s Ile Leu Gly Gln Leu     50              #     55              #     60 Gln Ala Ile Asn Lys 65 <210> SEQ ID NO 7 <211> LENGTH: 180 <212> TYPE: DNA<213> ORGANISM: ADENOVIRUS VR5 <400> SEQUENCE: 7atgtaagttt aataaagggt gagataatgt ttaacttgca tggcgtgtta aa#tccctttg     60atcttaatcc ctttgatctg gatccctttg atctccaacc ctttgatcta gt#cctatata    120atgcgccgtg ggctaatctt ggttacatct gacctcatgg aggcttggga gt#gtttggaa    180 <210> SEQ ID NO 8 <211> LENGTH: 314 <212> TYPE: DNA<213> ORGANISM: Homo sapiens <400> SEQUENCE: 8agcagctgcg ctgtcggggc caggccgggc tcccagtgga ttcgcgggca ca#gacgccca     60ggaccgcgct ccccacgtgg cggagggact ggggacccgg gcacccgtcc tg#ccccttca    120ccttccagct ccgcctcctc cgcgcggacc ccgccccgtc ccgacccctc cc#gggtcccc    180ggcccagccc cctccgggcc tcccagcccc cccctttcct ttccgcggcc cc#gccctctc    240ctcgcggcgc gagtttcagg cagcgctgcg tcctgctgcg cacgtgggaa gc#cctggccc    300 cggccacccc cgcg               #                  #                   #    314 <210> SEQ ID NO 9 <211> LENGTH: 22<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: Primer<400> SEQUENCE: 9 tgcattggta ccgtcatctc ta           #                   #                 22 <210> SEQ ID NO 10<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of Artificial #Sequence: Primer <400> SEQUENCE: 10 gttgctctgc ctctccactt            #                   #                   # 20 <210> SEQ ID NO 11<211> LENGTH: 41 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of Artificial #Sequence: Primer <400> SEQUENCE: 11cagatcaaag ggattaagat caaagggcca ttatgagcaa g     #                  #   41 <210> SEQ ID NO 12 <211> LENGTH: 43 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: Primer<400> SEQUENCE: 12 gatccctttg atctccaacc ctttgatcta gtccttaaga gtc    #                   # 43 <210> SEQ ID NO 13 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: Primer<400> SEQUENCE: 13 gggcgagtct ccacgtaaac g           #                   #                   #21 <210> SEQ ID NO 14<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of Artificial #Sequence: Primer <400> SEQUENCE: 14 gggcaccagc tcaatcagtc a           #                   #                   #21 <210> SEQ ID NO 15<211> LENGTH: 38 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of Artificial #Sequence: Primer <400> SEQUENCE: 15cggaattcaa gcttaattaa catcatcaat aatatacc       #                  #     38 <210> SEQ ID NO 16 <211> LENGTH: 31 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: Primer<400> SEQUENCE: 16 gcggctagcc accatggagc gaagaaaccc a        #                   #          31 <210> SEQ ID NO 17 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: Primer<400> SEQUENCE: 17 gccaccggta caacattcat t           #                   #                   #21 <210> SEQ ID NO 18<211> LENGTH: 40 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of Artificial #Sequence: Primer <400> SEQUENCE: 18agctgggctc tcttggtaca ccagtgcagc gggccaacta      #                  #    40 <210> SEQ ID NO 19 <211> LENGTH: 42 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: Primer<400> SEQUENCE: 19 cccaccactg tagtgctgcc aagagacgcc caggccgaag tt    #                   #  42 <210> SEQ ID NO 20 <211> LENGTH: 36<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: Primer<400> SEQUENCE: 20 ctgcgccccg ctattggtca tctgaacttc ggcctg      #                   #       36 <210> SEQ ID NO 21 <211> LENGTH: 32<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: Primer<400> SEQUENCE: 21 cttgcgggcg gctttagaca cagggtgcgg tc       #                   #          32 <210> SEQ ID NO 22 <211> LENGTH: 26<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: Primer<400> SEQUENCE: 22 cagatcaaag ggccattatg agcaag          #                   #              26 <210> SEQ ID NO 23<211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of Artificial #Sequence: Primer <400> SEQUENCE: 23gatccctttg atctagtcct taagagtc          #                  #             28 <210> SEQ ID NO 24 <211> LENGTH: 21 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: Primer<400> SEQUENCE: 24 atggcacaaa ctcctcaata a           #                   #                   #21 <210> SEQ ID NO 25<211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of Artificial #Sequence: Primer <400> SEQUENCE: 25 ccaagactac tcaacccgaa ta           #                   #                 22 <210> SEQ ID NO 26<211> LENGTH: 11 <212> TYPE: DNA <213> ORGANISM: Homo sapiens<400> SEQUENCE: 26 agatcaaagg g                #                  #                   #       11 <210> SEQ ID NO 27 <211> LENGTH: 35<212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 27gactagatca aagggatcca gatcaaaggg ccatt        #                  #       35 <210> SEQ ID NO 28 <211> LENGTH: 64 <212> TYPE: DNA<213> ORGANISM: Homo sapiens <400> SEQUENCE: 28gactagatca agggttggag atcaaaggga tccagatcaa agggattaag at#caaagggc     60 catt                  #                  #                   #             64 <210> SEQ ID NO 29 <211> LENGTH: 65<212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 29gactagatca aagggttgga gatcaaaggg atccagatca aagggattaa ga#tcaaaggg     60 ccatt                  #                  #                   #            65 <210> SEQ ID NO 30 <211> LENGTH: 48<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: Primer<400> SEQUENCE: 30tccctttgat ctccaaccct ttgatctagt cctatataat gcgccgtg  #                48 <210> SEQ ID NO 31 <211> LENGTH: 47 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: Primer<400> SEQUENCE: 31tccagatcaa agggattaag atcaaaggga tttaacacgc catgcaa   #                47 <210> SEQ ID NO 32 <211> LENGTH: 21 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: Taqman      oligo <400> SEQUENCE: 32 ttcgcttttg tgatacaggc a           #                   #                   #21 <210> SEQ ID NO 33<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of Artificial #Sequence: Taqman       oligo <400> SEQUENCE: 33gtcttggacg cgacgagaag             #                  #                   # 20 <210> SEQ ID NO 34 <211> LENGTH: 17<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: Taqman      oligo <400> SEQUENCE: 34 cggagcgttt gccgcgc             #                   #                   #   17 <210> SEQ ID NO 35<211> LENGTH: 17 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of Artificial #Sequence: Taqman       oligo <400> SEQUENCE: 35cggagcgttt gccgcgc              #                   #                  #   17 <210> SEQ ID NO 36 <211> LENGTH: 20 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: Taqman      oligo <400> SEQUENCE: 36 aacacctggt ccactgtcgc            #                   #                   # 20 <210> SEQ ID NO 37<211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of Artificial #Sequence: Taqman       oligo <400> SEQUENCE: 37ccgcgactcc gtttcaaccc aga            #                  #                23 <210> SEQ ID NO 38 <211> LENGTH: 21 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: Taqman      oligo <400> SEQUENCE: 38 tgcttccatc aaacgagttg g           #                   #                   #21 <210> SEQ ID NO 39<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of Artificial #Sequence: Taqman       oligo <400> SEQUENCE: 39gcgctgagtt tggctctagc             #                  #                   # 20 <210> SEQ ID NO 40 <211> LENGTH: 26<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: Taqman      oligo <400> SEQUENCE: 40 cggcggctgc tcaatctgta tcttca          #                   #              26 <210> SEQ ID NO 41<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of Artificial #Sequence: Taqman       oligo <400> SEQUENCE: 41ggttgattca tcggtcagtg c            #                  #                   #21 <210> SEQ ID NO 42 <211> LENGTH: 20<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: Taqman      oligo <400> SEQUENCE: 42 acgcctgcgg gtatgtattc            #                   #                   # 20 <210> SEQ ID NO 43<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of Artificial #Sequence: Taqman       oligo <400> SEQUENCE: 43aaaagcgacc gaaatagccc g            #                  #                   #21 <210> SEQ ID NO 44 <211> LENGTH: 23<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: Taqman      oligo <400> SEQUENCE: 44 tgatgtttga cgctacagcc ata           #                   #                23 <210> SEQ ID NO 45<211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of Artificial #Sequence: Taqman       oligo <400> SEQUENCE: 45gggatttgtg tttggtgcat tag            #                  #                23

What is claimed is:
 1. A viral DNA construct encoding for an adenoviruscapable of replication in a human or animal tumour cell and causingdeath of such tumour cells comprising one or more selected transcriptionfactor binding sites for a transcription factor the level or activity ofwhich is increased in a human or animal tumour cell relative to that ofa normal human or animal cell of the same type operatively positionedtogether with early viral protein gene open reading frames such as topromote expression of these open reading frames in the presence of saidselected transcription factor, the protein products of those openreading frames being mechanistically directly involved in viralconstruct nucleic acid replication selected from the group consisting ofviral polymerase, primase, nuclease, helicase, ligase, DNA terminalprotein and DNA binding protein and wherein the construct encodes anadenovirus E3 promoter which has in one or more mutations to nucleotideresidues which modify or delete transcription factor binding sites, asrelated to wild type adenovirus, selected from those in the NF1, NFKB,AP1 and ATF regions.
 2. A viral DNA construct as claimed in claim 1having a nucleic acid sequence corresponding to that of a wild typevirus sequence wherein it has one or more wild type transcription factorbinding sites replaced by one or more selected transcription factorbinding sites, these selected sites being operatively positioned in oneor more promoter regions which control expression of early genes such asto promote expression in the presence of said selected transcriptionfactor, the protein products of those genes being mechanisticallydirectly involved in viral nucleic acid replication and wherein theselected transcription, factor binding sites are for a transcriptionfactor the level or activity of which is increased in a human or animaltumour cell relative to that of a normal human or animal cell of thesame type.
 3. A viral DNA construct as claimed in claim 1 wherein theselected transcription factor binding sites are for a transcriptionfactor whose activity or level is specifically increased by causaloncogenic mutations.
 4. A construct as claimed in claim 1 wherein itsnucleic acid sequence, other than the selected sites, corresponds tothat of the genome of adenovirus Ad5, Ad40 or Ad41, or its nucleic acidsequence incorporates DNA encoding for fibre protein from Ad5, Ad40 orAd41, optionally with 15 to 25 lysines added to the end thereof.
 5. Aconstruct as claimed in claim 1 wherein the genes controlled by theselected transcription factor binding site are DNA polymerase, DNAterminal protein and/or DNA binding protein.
 6. A construct as claimedin claim 1 wherein its nucleic acid sequence corresponds to that of anadenovirus having a wild type E2 early promoter transcription factorbinding site replaced by the tumour cell specific transcription factorbinding site.
 7. A construct as claimed in claim 1 wherein it encodes afunctional viral RNA export capacity.
 8. A construct as claimed in claim1 having a nucleic acid sequence corresponding to that of an adenoviruswherein, in the E1 region, the E1B 55K gene is functional and/or intact.9. A construct as claimed in claim 2 wherein the selected transcriptionfactor binding site used in place of wild type site is selected fromTcf-4, RBPJK, Gli-I, HIF1alpha and telomerase promoter binding sites.10. A construct as claimed in claim 2 wherein the transcription factorbinding site that replaces the wild type site is selectively activatedin tumour cells containing oncogenic APC and β-catenin mutations.
 11. Aconstruct as claimed in claim 2 wherein the transcription factor bindingsite that replaces the wild type site is a single or multiple Tcf-4binding site sequence.
 12. A construct as claimed in claim 11 wherein itcomprises from 2 to 20 Tcf-4 binding site sequences at each replacedpromoter site.
 13. A construct as claimed in claim 1 wherein themutations reduce E2 gene transcription caused by E3 promoter activity.14. A construct as claimed in claim 1 wherein the mutations are silentmutations, being such as not to alter the predicted protein sequence ofany viral protein, but which alter the activity of one or more viralpromoters.
 15. A construct as claimed in claim 1 wherein its sequencecorresponds to that of an adenovirus genome wherein a region in the E2early promoter, which is not overlapped by coding sequence, is replacedwith multiple Tcf-4 binding site sequences.
 16. A construct as claimedin claim 1 wherein its sequence corresponds to that of an adenovirusgenome wherein the E2 late promoter en inactivated with silentmutations.
 17. A virus comprising or encoded by a DNA construct asclaimed in claim
 1. 18. A method of manufacture of a viral DNA constructas claimed in claim 2 comprising transforming a viral genome having oneor more wild type transcription factor binding sites controllingtranscription of genes having protein products directly mechanisticallyinvolved in viral nucleic acid replication to replace one or more ofthese by tumour specific transcription factor binding sites.
 19. Amethod as claimed in claim 18 wherein the viral genome is cloned by gaprepair in a circular YAC/BAC in yeast.
 20. A method as claimed in claim18 wherein the genome is modified by two step gene replacement.
 21. Amethod as claimed in claim 18 wherein the modified genome is transferredto a prokaryote for production of viral construct DNA.
 22. A method ofmanufacture of a virus comprising transferring viral construct DNAproduced by a method as claimed in claim 18 to a mammalian cell forproduction of virus.
 23. A method for treating a patient suffering fromneoplasms comprising causing a viral DNA construct or virus as claimedin claim 1 to infect tissues of the patient, including or restricted tothose of the neoplasm, and allowing replication such that neoplasm cellsare caused to be killed.